Blockade of Brain Stem Gap Junctions Increases Phrenic Burst Frequency and Reduces Phrenic Burst Synchronization in Adult Rat

Abstract

Recent investigations have examined the influence of gap junctional communication on generation and modulation of respiratory rhythm and inspiratory motoneuron synchronization in vitro using transverse medullary slice and en bloc brain stem-spinal cord preparations obtained from neonatal (1–5 days postnatal) mice. Gap junction proteins, however, have been identified in both neurons and glia in brain stem regions implicated in respiratory control in both neonatal and adult rodents. Here, we used an in vitro arterially perfused rat preparation to examine the role of gap junctional communication on generation and modulation of respiratory rhythm and inspiratory motoneuron synchronization in adult rodents. We recorded rhythmic inspiratory motor activity from one or both phrenic nerves before and during pharmacological blockade (i.e., uncoupling) of brain stem gap junctions using carbenoxolone (100 μM), 18α-glycyrrhetinic acid (25–100 μM), 18β-glycyrrhetinic acid (25–100 μM), octanol (200–300 μM), or heptanol (200 μM). During perfusion with a gap junction uncoupling agent, we observed an increase in the frequency of phrenic bursts (∼95% above baseline frequency; P < 0.001) and a decrease in peak amplitude of integrated phrenic nerve discharge ( P < 0.001). The increase in frequency of phrenic bursts resulted from a decrease in both T I ( P < 0.01) and T E ( P < 0.01). In addition, the pattern of phrenic nerve discharge shifted from an augmenting discharge pattern to a “bell-shaped” or square-wave discharge pattern in most experiments. Spectral analyses using a fast Fourier transform (FFT) algorithm revealed a reduction in the peak power of both the 40- to 50-Hz peak (corresponding to the MFO) and 90- to 110-Hz peak (corresponding to the HFO) although spurious higher frequency activity (≥130 Hz) was observed, suggesting an overall loss or reduction in inspiratory-phase synchronization. Although additional experiments are required to identify the specific brain stem regions and cell types (i.e., neurons, glia) mediating the observed modulations in phrenic motor output, these findings suggest that gap junction communication modulates generation of respiratory rhythm and inspiratory motoneuron synchronization in adult rodents in vitro.

Effects of heptanol and carbenoxolone on noradrenaline induced contractions in guinea pig vas deferens

“…Two putative gap junction blockers which have been used widely in the assessment of syncytial function are the alkanol, 1-heptanol, and the hydroxysteroid dehydrogenase inhibitor, carbenoxolone. Recent reports have suggested that carbenoxolone over a concentration range of 10-200 μM blocks gap junctions more selectively than does heptanol , which has been suggested to have non-specific actions on ionic channels (Margineanu and Klitgaard, 2001;Solomon et al, 2003). In the present study we find, in the smooth muscle of GPVD, that the effects of carbenoxolone are indeed more consistent with gap junction block than are those of heptanol.…”

Comparison of mathematical models of fast oscillatory rhythms in inspiratory discharges

“…The phrenic discharge data used for this study were obtained in our previous experiments conducted in an in vitro arterially-perfused decerebrate adult rat (81-125g, ~5-6 wk old; n=10) preparation [4].…”

“…Two prominent peaks in the PSD, referred to as medium and high frequency oscillations (MFO and HFO, respectively), have been identified, and they are believed to represent synchronization of inspiratory neuronal activities [1,3,4]. We have recently developed a 9 parameter mathematical model that is capable of characterizing the major spectral features associated with these inspiratory-phase fast oscillations [5].…”

Ventilatory effects of gap junction blockade in the RTN in awake rats

“…GZA is a congener of carbenoxolone, and it has been used as a control substance to test for nonspecific effects of carbenoxolone (46,63). We studied the effect of unilateral perfusion of GZA in the RTN on ventilatory responses to CO 2 in five animals (mean age 10.2 Ϯ 0.9 wk).…”

“…Immunohistochemical staining demonstrated the presence of Cx26, Cx32, and Cx36 in multiple regions of the brain stem involved in respiratory control (62,64). Of the respiratory-related sites in which gap junctions are expressed, the pre-Bötzinger complex, locus ceruleus (LC), retrotrapezoid nucleus (RTN) in the ventrolateral medulla, and nucleus of the solitary tract (NTS) are sensitive to CO 2 (9,44,63). Therefore, gap junctions may be a necessary component of central CO 2 chemosensitivity (12).…”